1. Field of the Invention
The invention relates to particle analyzers wherein electronic impedance measurements and optical measurements are made on particles entrained in a liquid stream.
2. Description of the Prior Art
Since its conception more than 25 years ago, the principle of particle counting and sizing invented by Wallace H. Coulter has resulted in numerous methods and apparatuses for the electronic counting, sizing, and analysis of microscopic particles, which are scanned in a fluid suspension, as shown by the pioneer U.S. Pat. No. 2,656,508 to Coulter. In this prior art arrangement, a D.C. electric current flow is established between two vessels by suspending electrodes in the respective bodies of the suspension fluid. The only fluid connection between the two bodies is through an orifice; hence, an electric current flow and field are established in the orifice. The orifice and the resultant electric field in and around it constitute a sensing zone. As each particle passes through the sensing zone, for the duration of the passage, the impedance of the contents of the sensing zone will change, thereby modulating the electric current flow and electric field in the sensing zone, and hence causing the generation of a signal to be applied to a detector suitably arranged to respond to such change. (The mark "Coulter" is a registered trademark, Registration No. 995,825, of Coulter Electronics, Inc. of Hialeah, Fla.) The heretofore described particle analyzers will be referred to as "impedance sensing particle analyzers".
Numerous particle analyzers have been developed wherein optical measurements, such as scattered light detection, fluorescent light detection, and light absorbance detection, are made on an entrained stream of particles. These particle analyzers will be hereinafter referred to as "optical particle analyzers". The most advanced state of the art for optical particle analyzers is shown in U.S. Pat. Nos. 4,188,542, 4,188,543, 4,189,236 and 4,199,686, the assignee therein being the same as in the present invention. Each of these patents are incorporated by reference herein. Each of these optical particle analyzers is directed toward using reflector optics for collecting large solid angle optical signals emanating from a sensing zone whereat illuminating radiation impinges upon the stream of particles. The use of these wide angle collectors has resulted in greater light collection efficiency, better optical and hence electronic signal to noise ratios and the minimizing of the effects of preferential signal emissions in unpredictable directions. The improved sensitivity in detecting characteristics of biological cells, resulting from the utilization of these wide angle collectors, has opened new areas of cell analysis that heretofore were not available.
As can be seen from the prior art, the development of optical particle analyzers and impedance sensing particle analyzers have proceeded along different paths, without the two types of particle measurements being combined in a single apparatus. An impedance sensing orifice has been used in combination with downstream light absorbance detection, scattered light detection and fluorescent light detection, as disclosed in U.S. Pat. No. 3,710,933 to Fulwyler et al. U.S. Pat. No. 3,710,933 is incorporated by reference herein. However, the types of optical measurements made downstream from the orifice do not provide the type and quality of information, nor the sensitivity of detection, required for the hereinafter described invention.
In the Fulwyler particle analyzer of U.S. Pat. No. 3,710,933 and in the prior art apparatuses modeled thereafter, a pair of concentric tubes have been used, one tube for introducing the sample of suspended particles and the other tube for providing a first liquid sheath around the sample. The liquid sheath hydrodynamically focuses the particles as they pass through an orifice disc, which is mounted at the end of the sheath tube and has an impedance sensing orifice formed therein. By virtue of this arrangement, a second liquid sheath is required for hydrodynamically focusing the particles as they proceed from the orifice, through the optical sensing zone, into an exit tube or nozzle. This has been the accepted and only known way of accomplishing hydrodynamically focused movement of the particles through both an impedance sensing zone and a subsequent optical sensing zone for most of the last decade.
In addition to the optical disadvantages previously discussed, the above described Fulwyler particle analyzer has several other disadvantages. First, as shown by the design of the Fulwyler analyzer, it has always been assumed in accepted prior art practices, that the sensing orifice must be positioned upstream of the optical sensing zone. The sheath tube and orifice disc containing the sensing orifice must be made sufficiently large to provide structural strength. On the other hand, the particles must be very accurately aligned and hydrodynamically focused to pass through the relatively small optical sensing zone. Typically, a laser beam has a cross-sectional Gaussian intensity profile, and the particles must each pass through the center of the profile to be substantially uniformly irradiated at maximum intensities. Hence, the closer the end of the sheath tube is positioned to the optical sensing zone, the better the alignment of the particles through the optical sensing zone and therefore the better the optical signal resolution. Unfortunately, as will be seen in the hereinafter described invention, if light is to be collected in a nearly 4 solid angle, the large end of the sheath tube blocks a substantial portion of the light, if it is positioned in close proximity to the optical sensing zone, as is the case with the Fulwyler analyzer. Therefore, this prior art analyzer design creates a dilemna of having an undesirable tradeoff between having to sacrifice optical signal resolution or light collection efficiency or having to settle upon a less than satisfactory combination to minimize the two design problems.